Recent and earlier studies suggest a direct link between obesity, a major cause of morbidity and mortality, sweet taste, and craving for sweet. This craving is generally satisfied with sugars, which are still overwhelmingly preferred, despite availability of a number of non-caloric sugar substitutes. A likely reason for this is that no substitute tastes like sucrose. We predict that there will be none until the molecular mechanism(s) of sweet taste is fully understood. Here we propose to combine the easily modified, high potency sweet plant protein, Brazzein, which we discovered (Ming and Hellekant 1994), with the recently discovered family of transmembrane receptors for sweet taste T1Rs to achieve insight into the peripheral mechanism(s) of sweet taste. This knowledge can then be used to create better non-caloric sugar substitutes and will ultimately serve the clinic. The experimental goals of this proposal are: 1. To generate brazzein mutants for structure-function analyses: a. To design and produce brazzein mutants, b. To examine the structurat and dynamic consequences of the mutations. 2. To characterize the brazzein-taste receptor interaction in vitro and in vivo. a). To test brazzein mutants for direct binding and activity assay with T1R2/T1R3 sweet receptors; b). To record the effects of brazzein mutations on responses from rhesus monkey in single chorda tympani and glossopharyngeal taste fibers, c). To determine the sweetness of the brazzein mutants psychophysically with human subjects. 3. To model the brazzein-taste receptor interaction, a). To homology-model human T1R2/T1R3 extra-cellular domain heterodimers, b). To evaluate possible docking sites for brazzein and brazzein mutants on these extra cellular domain heterodimers. Ming, D. and G. Hellekant (1994). "Brazzein, a new high-potency thermostable sweet protein from Pentadiplandra brazzeana B." FEBS Letters 355(1): 106-8.